APPLICANT'S DESCRIPTION: The major spliceosomal snRNAs U1, U2, U4, U5, and U6 share common features of post-transcriptional modification, including 5' cap methylation and internal pseudouridylation and 2'-O-methylation. The modified nucleotides in these snRNAs are remarkably conserved from species to species, strongly suggesting that they are biologically significant. The long-range goal of this project is to understand the underlying biological principles of spliceosomal snRNA modifications. Detailed investigation of this process has been impeded by the lack of effective assays and experimental systems. Recently, our lab and others developed several highly sensitive assays for detecting modified nucleotides in RNA. Using Xenopus oocytes, we also developed an experimental system for performing extremely efficient modifications in spliceosomal snRNAs. For the first time, these two developments provide us with tools to address the fundamental questions regarding spliceosomal snRNA modification. Because it is the most extensively modified, U2 is the focus of our study. There are three major goals in this proposal. (1) To identify which modified nucleotides are required for U2 activity, and how these nucleotides contribute to U2 function. We will generate a variety of U2 constructs in which selectively modified or un-modifiable nucleotides are introduced. The ability of these U2 derivatives to reconstitute U2 snRNP assembly and participate in splicing will be tested and important modified nucleotides will be precisely determined. These critical modified nucleotides will then be targeted for 4-thioU site-specific cross-linking analysis. (2) To address the subcellular localization of the machinery responsible for U2 internal modifications. We will use a systematic mutational analysis to identify key sequence elements in U2 that are required for nucleotide modification and for proper subcompartmental targeting as monitored by fluorescence immunocytochemistry. Mutant U2 RNAs lacking these critical sequence elements will then be attached to various signal sequences in order to target our experimental U2 RNAs to desired subcellular compartments. Restoration of modification in U2 mutants will provide valuable information as to where U2 internal modifications occur. (3) To identify the cellular machinery involved in U2 modification. Our preliminary results suggest that 2'-O-methylation of U2 is mediated by nuclear guide RNAs, in a manner similar to that for rRNAs. We will use a variety of biochemical approaches to search for such putative guide RNAs and to identify the modifying activity responsible for 2'-O-methylation and pseudouridylation.